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Abstract:

The invention relates to the quantitative measurement of steroidal
compounds by mass spectrometry. In a particular aspect, the invention
relates to methods for quantitative measurement of steroidal compounds
from multiple samples by mass spectrometry.

Claims:

1. A method for determining the amount of a steroidal compound in each of
a plurality of test samples with a single mass spectrometric assay,
wherein the steroidal compound prior to processing is the same in each
test sample, the method comprising: processing a plurality of test
samples differently to form a plurality of processed samples, wherein
said processing comprises subjecting each test sample to a different
Cookson-type derivatizing agent under conditions suitable to generate
Cookson-type derivatized steroidal compounds, and wherein as a result of
said processing, the steroidal compound in each processed sample is
distinguishable by mass spectrometry from the steroidal compound in other
processed samples; combining the processed samples to form a multiplex
sample; subjecting the multiplex sample to an ionization source under
conditions suitable to generate one or more ions detectable by mass
spectrometry, wherein one or more ions generated from the steroidal
compound from each processed sample are distinct from one or more ions
from the steroidal compound from the other processed samples; detecting
the amount of one or more ions from the steroidal compound from each
processed sample by mass spectrometry; and relating the amount of one or
more ions from the steroidal compound from each processed sample to the
amount of the steroidal compound in each test sample.

2. The method of claim 1, wherein said plurality of processed samples
comprises one processed sample with underivatized steroidal compounds.

3. The method of claim 1, wherein said different derivatizing agents are
isotopic variants of each another.

6. The method of claims claim 1, wherein the plurality of samples
comprises two samples, a first derivatizing reagent is
4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and a second derivatizing
reagent is 13C6-4-phenyl-1,2,4-triazoline-3,5-dione
(13C6-PTAD).

7. The method of claim 1, wherein said steroidal compound is a vitamin D
or vitamin D related compound.

Description:

FIELD OF THE INVENTION

[0001] The invention relates to the quantitative measurement of steroidal
compounds by mass spectrometry. In a particular aspect, the invention
relates to methods for quantitative measurement of steroidal compounds
from multiple samples by mass spectrometry.

BACKGROUND OF THE INVENTION

[0002] Steroidal compounds are any of numerous naturally occurring or
synthetic fat-soluble organic compounds having as a basis 17 carbon atoms
arranged in four rings and including the sterols and bile acids, adrenal
and sex hormones, certain natural drugs such as digitalis compounds, as
well as certain vitamins and related compounds (such as vitamin D,
vitamin D analogues, and vitamin D metabolites).

[0003] Many steroidal compounds are biologically important. For example,
vitamin D is an essential nutrient with important physiological roles in
the positive regulation of calcium (Ca2+) homeostasis. Vitamin D can
be made de novo in the skin by exposure to sunlight or it can be absorbed
from the diet. There are two forms of vitamin D; vitamin D2
(ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D3
is the form synthesized de novo by animals. It is also a common
supplement added to milk products and certain food products produced in
the United States. Both dietary and intrinsically synthesized vitamin
D3 must undergo metabolic activation to generate the bioactive
metabolites. In humans, the initial step of vitamin D3 activation
occurs primarily in the liver and involves hydroxylation to form the
intermediate metabolite 25-hydroxycholecalciferol (calcifediol;
25OHD3). Calcifediol is the major form of Vitamin D3 in
circulation. Circulating 25OHD3 is then converted by the kidney to
form 1,25-dihydroxyvitamin D3 (calcitriol; 1,25(OH)2D3),
which is generally believed to be the metabolite of Vitamin D3 with
the highest biological activity.

[0004] Vitamin D2 is derived from fungal and plant sources. Many
over-the-counter dietary supplements contain ergocalciferol (vitamin
D2) rather than cholecalciferol (vitamin D3). Drisdol, the only
high-potency prescription form of vitamin D available in the United
States, is formulated with ergocalciferol. Vitamin D2 undergoes a
similar pathway of metabolic activation in humans as Vitamin D3,
forming the metabolites 25OHD2 and 1,25(OH)2D2. Vitamin
D2 and vitamin D3 have long been assumed to be biologically
equivalent in humans, however recent reports suggest that there may be
differences in the bioactivity and bioavailability of these two forms of
vitamin D (Armas et. al., (2004) J. Clin. Endocrinol. Metab.
89:5387-5391).

[0005] Measurement of vitamin D, the inactive vitamin D precursor, is rare
in clinical settings. Rather, serum levels of 25-hydroxyvitamin D3,
25-hydroxyvitamin D2, and total 25-hydroxyvitamin D ("25OHD") are
useful indices of vitamin D nutritional status and the efficacy of
certain vitamin D analogs. The measurement of 25OHD is commonly used in
the diagnosis and management of disorders of calcium metabolism. In this
respect, low levels of 25OHD are indicative of vitamin D deficiency
associated with diseases such as hypocalcemia, hypophosphatemia,
secondary hyperparathyroidism, elevated alkaline phosphatase,
osteomalacia in adults and rickets in children. In patients suspected of
vitamin D intoxication, elevated levels of 25OHD distinguishes this
disorder from other disorders that cause hypercalcemia.

[0006] Measurement of 1,25(OH)2D is also used in clinical settings.
Certain disease states can be reflected by circulating levels of
1,25(OH)2D, for example kidney disease and kidney failure often
result in low levels of 1,25(OH)2D. Elevated levels of
1,25(OH)2D may be indicative of excess parathyroid hormone or can be
indicative of certain diseases such as sarcoidosis or certain types of
lymphomas.

[0008] The present invention provides methods for detecting the amount of
a steroidal compound in each of a plurality of test samples with a single
mass spectrometric assay. The methods include processing each test sample
differently to form a plurality of processed samples, wherein as a result
of the processing, the steroidal compound in each processed sample is
distinguishable by mass spectrometry from the steroidal compound in other
processed samples; combining the processed samples to form a multiplex
sample; subjecting the multiplex sample to an ionization source under
conditions suitable to generate one or more ions detectable by mass
spectrometry, wherein one or more ions generated from the steroidal
compound from each processed sample are distinct from one or more ions
from the steroidal compound from the other processed samples; detecting
the amount of one or more ions from the steroidal compound from each
processed sample by mass spectrometry; and relating the amount of one or
more ions from the steroidal compound from each processed sample to the
amount of the steroidal compound in each test sample.

[0009] In some embodiments, processing a test sample comprises subjecting
each test sample to a different derivatizing agent under conditions
suitable to generate derivatized steroidal compounds. In some embodiment,
one test sample may be processed without subjecting the sample to a
derivatizing agent.

[0010] In some embodiments, the different derivatizing agents used in the
processing of the plurality of test samples are isotopic variants of each
another. In some embodiments, the different derivatizing agents are
Cookson-type derivatizing agents; such as Cookson-type derivatization
agents selected from the group consisting of
4-phenyl-1,2,4-triazoline-3,5-dione (PTAD),
4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-tri-
azoline-3,5-dione (DMEQTAD), 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione
(NPTAD), 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), and
isotopic variants thereof. In one related embodiment, the Cookson-type
derivatizing agents are isotopic variants of
4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). In a specific embodiment, the
plurality of samples comprises two samples, a first Cookson-type
derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and a
second Cookson-type derivatizing reagent is
13C6-4-phenyl-1,2,4-triazoline-3,5-dione (13C6-PTAD).

[0011] In some embodiments, the steroidal compound is a vitamin D or
vitamin D related compound. In related embodiments, the steroidal
compound is selected from the group consisting of vitamin D2,
vitamin D3, 25-hydroxyvitamin D2 (25OHD2),
25-hydroxyvitamin D3 (25OHD3), 1α,25-dihydroxyvitamin
D2 (1α,25OHD2), and 1α,25-dihydroxyvitamin D3
(1α,25OHD3). In a specific embodiment, the steroidal compound
is 25-hydroxyvitamin D2 (25OHD2) or 25-hydroxyvitamin D3
(25OHD3).

[0012] The methods described above may be conducted for the analysis of
two or more steroidal compounds in each of a plurality of test samples.
In some of these embodiments, the two or more steroidal compounds in each
test sample may include at least one steroidal compound selected from the
group consisting of 25-hydroxyvitamin D2 (25OHD2) and
25-hydroxyvitamin D3 (25OHD3). In some embodiments, the two or
more steroidal compounds in each test sample are 25-hydroxyvitamin
D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3).

[0013] In specific embodiments, the amount of one or more vitamin D or
vitamin D related compounds in each of two test samples is determined
with a single mass spectrometric assay. In this embodiment, a first
processed sample is generated by subjecting a first test sample to a
first isotopic variant of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD)
under conditions suitable to generate one or more vitamin D or vitamin D
related derivatives; a second processed sample is generated by subjecting
a second test sample to a second isotopic variant of
4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) under conditions suitable to
generate one or more vitamin D or vitamin D related derivatives, wherein
the first and second isotopic variant of PTAD are distinguishable by mass
spectrometry; the first processed sample is mixed with the second
processed sample to form a multiplex sample; one or more vitamin D or
vitamin D related derivatives from each processed sample in the multiplex
sample are subjected to an ionization source under conditions suitable to
generate one or more ions detectable by mass spectrometry, wherein one or
more ions from each vitamin D or vitamin D related derivatives from the
first processed sample are distinct from the one or more ions from
vitamin D or vitamin D related derivatives from the second processed
sample; the amounts of one or more ions from one or more vitamin D or
vitamin D related derivatives from each processed sample are determined
by mass spectrometry; and the amounts of the determined ions are related
to the amounts of vitamin D or vitamin D related compound in the first or
second test sample.

[0014] In some specific embodiments, the first isotopic variant of
4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is
4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and the second isotopic
variant of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is
13C6-4-phenyl-1,2,4-triazoline-3,5-dione (13C6-PTAD).

[0015] In some specific embodiments, the one or more vitamin D or vitamin
D related compounds are selected from the group consisting of
25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3
(25OHD3). In some related specific embodiments, the one or more
vitamin D or vitamin D related compounds include 25-hydroxyvitamin
D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3). In
some related specific embodiments, the one or more vitamin D or vitamin D
related compounds are 25-hydroxyvitamin D2 (25OHD2) and
25-hydroxyvitamin D3 (25OHD3).

[0016] In some embodiments, the multiplex sample is subjected to an
extraction column and an analytical column prior to being subjected to an
ionization source. In some related embodiments, the extraction column is
a solid-phase extraction (SPE) column. In other related embodiments, the
extraction column is a turbulent flow liquid chromatography (TFLC)
column. In some embodiments, the analytical column is a high performance
liquid chromatography (HPLC) column.

[0017] In embodiments which utilize two or more of an extraction column,
an analytical column, and an ionization source, two or more of these
components may be connected in an on-line fashion to allow for automated
sample processing and analysis.

[0018] In the methods described herein, mass spectrometry may be tandem
mass spectrometry. In embodiments utilizing tandem mass spectrometry,
tandem mass spectrometry may be conducted by any method known in the art,
including for example, multiple reaction monitoring, precursor ion
scanning, or product ion scanning.

[0019] In the methods described herein, steroidal compounds may be ionized
by any suitable ionization technique known in the art. In some
embodiments, the ionization source is a laser diode thermal desorption
(LDTD) ionization source.

[0020] In preferred embodiments, the test samples comprise biological
samples, such as plasma or serum.

[0021] As used herein, the term "multiplex sample" refers to a sample
prepared by pooling two or more samples to form the single "multiplex"
sample which is then subject to mass spectrometric analysis. In the
methods described herein, two or more test samples are each processed
differently to generate multiple differently processed samples. These
multiple differently processed samples are then pooled to generate a
single "multiplex" sample, which is then subject to mass spectrometric
analysis.

[0022] As used herein, the term "steroidal compound" refers to any of
numerous naturally occurring or synthetic fat-soluble organic compounds
having as a basis 17 carbon atoms arranged in four rings and including
the sterols and bile acids, adrenal and sex hormones, certain natural
drugs such as digitalis compounds, as well as certain vitamins and
related compounds (such as vitamin D, vitamin D analogues, and vitamin D
metabolites).

[0023] As used herein, the term "vitamin D or vitamin D related compound"
refers to any natural or synthetic form of vitamin D, or any chemical
species related to vitamin D generated by a transformation of vitamin D,
such as intermediates and products of vitamin D metabolism. For example,
vitamin D may refer to one or more of vitamin D2 and vitamin
D3. Vitamin D may also be referred to as "nutritional" vitamin D to
distinguish from chemical species generated by a transformation of
vitamin D. Vitamin D related compounds may include chemical species
generated by biotransformation of analogs of, or a chemical species
related to, vitamin D2 or vitamin D3. Vitamin D related
compounds, specifically vitamin D metabolites, may be found in the
circulation of an animal and/or may be generated by a biological
organism, such as an animal. Vitamin D metabolites may be metabolites of
naturally occurring forms of vitamin D or may be metabolites of synthetic
vitamin D analogs. In certain embodiments, vitamin D related compounds
may include one or more vitamin D metabolites selected from the group
consisting of 25-hydroxyvitamin D3, 25-hydroxyvitamin D2,
1α,25-dihydroxyvitamin D3 and 1α,25-dihydroxyvitamin
D2.

[0024] As used herein, "derivatizing" means reacting two molecules to form
a new molecule. Thus, a derivatizing agent is an agent that is reacted
with another substance to derivatize the substance. For example,
4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is a derivatizing reagent that
may be reacted with a vitamin D metabolite to form a PTAD-derivatized
vitamin D metabolite.

[0025] As used herein, "different derivatizing agents" are derivatizing
agents that are distinguishable by mass spectrometry. As one example, two
isotopic variants of the same derivatizing agent are distinguishable by
mass spectrometry. As another example, halogenated variants of the same
derivatizing agent are also distinguishable by mass spectrometry. For
example, halogenated and non-halogenated versions of the same
Cookson-type agent, such as 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD),
may be used. Further, two halogenated versions of the same Cookson-type
agent, but halogenated with different halogens or with different numbers
of halogens, may be used. As another example, two different Cookson-type
agents, such as 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD),
4-methyl-1,2,4-triazoline-3,5-dione (MTAD), and
4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD), may be used. The
above examples illustrate the principle of derivatizing agents that are
distinguishable by mass spectrometry. Other examples, including
combinations of any of the above, may be possible as would be appreciated
by one of skill in the art.

[0026] As used herein, the names of derivatized forms of steroidal
compounds include an indication as to the nature of derivatization. For
example, the PTAD derivative of 25-hydroxyvitamin D2 is indicated as
PTAD-25-hydroxyvitamin D2 (or PTAD-25OHD2).

[0028] In certain preferred embodiments of the methods disclosed herein,
mass spectrometry is performed in positive ion mode. Alternatively, mass
spectrometry is performed in negative ion mode. Various ionization
sources, including for example atmospheric pressure chemical ionization
(APCI) or electrospray ionization (ESI), may be used in embodiments of
the present invention. In certain embodiments, steroidal compounds,
including vitamin D and vitamin D related compounds, are measured using
APCI in positive ion mode.

[0029] In preferred embodiments, one or more separately detectable
internal standards are provided in the sample, the amount of which are
also determined in the sample. In these embodiments, all or a portion of
both the analyte(s) of interest and the internal standard(s) present in
the sample are ionized to produce a plurality of ions detectable in a
mass spectrometer, and one or more ions produced from each are detected
by mass spectrometry. Exemplary internal standard(s) include vitamin
D2-[6, 19, 19]-2H3, vitamin D2-[24, 24, 24, 25, 25,
25]-2H6, vitamin D3-[6, 19, 19]-2H3, vitamin
D3-[24, 24, 24, 25, 25, 25]-2H6, 25OHD2-[6, 19,
19]-2H3, 25OHD2-[24, 24, 24, 25, 25, 25]-2H6,
25OHD3-[6, 19, 19]-2H3, 25OHD3-[24, 24, 24, 25, 25,
25]-2H6, 1α,25OHD2-[6, 19, 19]-2H3,
1α,25OHD2-[24, 24, 24, 25, 25, 25]-2H6,
1α,25OHD3-[6, 19, 19]-2H3, 1α,25OHD3-[24,
24, 24, 25, 25, 25]-2H6.

[0030] One or more separately detectable internal standards may be
provided in the sample prior to treatment of the sample with a
Cookson-type derivatizing reagent. In these embodiments, the one or more
internal standards may undergo derivatization along with the endogenous
steroidal compounds, in which case ions of the derivatized internal
standards are detected by mass spectrometry. In these embodiments, the
presence or amount of ions generated from the analyte of interest may be
related to the presence of amount of analyte of interest in the sample.
In some embodiments, the internal standards may be isotopically labeled
versions of steroidal compounds under investigation. For example in an
assay where vitamin D metabolites are analytes of interest,
25OHD2-[6, 19, 19]-2H3 or 25OHD3-[6, 19,
19]-2H3 may be used as an internal standard. In embodiments
where 25OHD2-[6, 19, 19]-2H3 is used as internal
standards, PTAD-25OHD2-[6, 19, 19]-2H3 ions detectable in
a mass spectrometer are selected from the group consisting of positive
ions with a mass/charge ratio (m/z) of 573.30±0.50 and 301.10±0.50.
In related embodiments, a PTAD-25OHD2-[6, 19, 19]-2H3
precursor ion has a m/z of 573.30±0.50, and a fragment ion has m/z of
301.10±0.50. In embodiments where 25OHD3-[6, 19,
19]-2H3 is used as an internal standard, PTAD-25OHD3-[6,
19, 19] ions detectable in a mass spectrometer are selected from the
group consisting of positive ions with a mass/charge ratio (m/z) of
561.30±0.50 and 301.10±0.50. In related embodiments, a
PTAD-25OHD3-[6, 19, 19] precursor ion has a m/z of 561.30±0.50,
and a fragment ion has m/z of 301.10±0.50.

[0031] As used herein, an "isotopic label" produces a mass shift in the
labeled molecule relative to the unlabeled molecule when analyzed by mass
spectrometric techniques. Examples of suitable labels include deuterium
(2H), 13C, and 15N. For example, 25OHD2-[6, 19, 19]
and 25OHD3-[6, 19, 19] have masses about 3 mass units higher than
25OHD2 and 25OHD3. The isotopic label can be incorporated at
one or more positions in the molecule and one or more kinds of isotopic
labels can be used on the same isotopically labeled molecule.

[0032] In other embodiments, the amount of the vitamin D metabolite ion or
ions may be determined by comparison to one or more external reference
standards. Exemplary external reference standards include blank plasma or
serum spiked with one or more of 25OHD2, 25OHD2-[6, 19, 19],
25OHD3, and 25OHD3-[6, 19, 19]. External standards typically
will undergo the same treatment and analysis as any other sample to be
analyzed, including treatment with one or more Cookson-type reagents
prior to mass spectrometry.

[0033] In certain preferred embodiments, the limit of quantitation (LOQ)
of 25OHD2 is within the range of 1.9 ng/mL to 10 ng/mL, inclusive;
preferably within the range of 1.9 ng/mL to 5 ng/mL, inclusive;
preferably about 1.9 ng/mL. In certain preferred embodiments, the limit
of quantitation (LOQ) of 25OHD3 is within the range of 3.3 ng/mL to
10 ng/mL, inclusive; preferably within the range of 3.3 ng/mL to 5 ng/mL,
inclusive; preferably about 3.3 ng/mL.

[0034] As used herein, unless otherwise stated, the singular forms "a,"
"an," and "the" include plural reference. Thus, for example, a reference
to "a protein" includes a plurality of protein molecules.

[0035] As used herein, the term "purification" or "purifying" does not
refer to removing all materials from the sample other than the analyte(s)
of interest. Instead, purification refers to a procedure that enriches
the amount of one or more analytes of interest relative to other
components in the sample that may interfere with detection of the analyte
of interest. Purification of the sample by various means may allow
relative reduction of one or more interfering substances, e.g., one or
more substances that may or may not interfere with the detection of
selected parent or daughter ions by mass spectrometry. Relative reduction
as this term is used does not require that any substance, present with
the analyte of interest in the material to be purified, is entirely
removed by purification.

[0036] As used herein, the term "solid phase extraction" or "SPE" refers
to a process in which a chemical mixture is separated into components as
a result of the affinity of components dissolved or suspended in a
solution (i.e., mobile phase) for a solid through or around which the
solution is passed (i.e., solid phase). In some instances, as the mobile
phase passes through or around the solid phase, undesired components of
the mobile phase may be retained by the solid phase resulting in a
purification of the analyte in the mobile phase. In other instances, the
analyte may be retained by the solid phase, allowing undesired components
of the mobile phase to pass through or around the solid phase. In these
instances, a second mobile phase is then used to elute the retained
analyte off of the solid phase for further processing or analysis. SPE,
including TFLC, may operate via a unitary or mixed mode mechanism. Mixed
mode mechanisms utilize ion exchange and hydrophobic retention in the
same column; for example, the solid phase of a mixed-mode SPE column may
exhibit strong anion exchange and hydrophobic retention; or may exhibit
column exhibit strong cation exchange and hydrophobic retention.

[0037] As used herein, the term "chromatography" refers to a process in
which a chemical mixture carried by a liquid or gas is separated into
components as a result of differential distribution of the chemical
entities as they flow around or over a stationary liquid or solid phase.

[0038] As used herein, the term "liquid chromatography" or "LC" means a
process of selective retardation of one or more components of a fluid
solution as the fluid uniformly percolates through a column of a finely
divided substance, or through capillary passageways. The retardation
results from the distribution of the components of the mixture between
one or more stationary phases and the bulk fluid, (i.e., mobile phase),
as this fluid moves relative to the stationary phase(s). Examples of
"liquid chromatography" include reverse phase liquid chromatography
(RPLC), high performance liquid chromatography (HPLC), and turbulent flow
liquid chromatography (TFLC) (sometimes known as high turbulence liquid
chromatography (HTLC) or high throughput liquid chromatography).

[0039] As used herein, the term "high performance liquid chromatography"
or "HPLC" (sometimes known as "high pressure liquid chromatography")
refers to liquid chromatography in which the degree of separation is
increased by forcing the mobile phase under pressure through a stationary
phase, typically a densely packed column.

[0040] As used herein, the term "turbulent flow liquid chromatography" or
"TFLC" (sometimes known as high turbulence liquid chromatography or high
throughput liquid chromatography) refers to a form of chromatography that
utilizes turbulent flow of the material being assayed through the column
packing as the basis for performing the separation. TFLC has been applied
in the preparation of samples containing two unnamed drugs prior to
analysis by mass spectrometry. See, e.g., Zimmer et al., J Chromatogr A
854: 23-35 (1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368,
5,795,469, and 5,772,874, which further explain TFLC. Persons of ordinary
skill in the art understand "turbulent flow". When fluid flows slowly and
smoothly, the flow is called "laminar flow". For example, fluid moving
through an HPLC column at low flow rates is laminar. In laminar flow the
motion of the particles of fluid is orderly with particles moving
generally in straight lines. At faster velocities, the inertia of the
water overcomes fluid frictional forces and turbulent flow results. Fluid
not in contact with the irregular boundary "outruns" that which is slowed
by friction or deflected by an uneven surface. When a fluid is flowing
turbulently, it flows in eddies and whirls (or vortices), with more
"drag" than when the flow is laminar. Many references are available for
assisting in determining when fluid flow is laminar or turbulent (e.g.,
Turbulent Flow Analysis: Measurement and Prediction, P. S. Bernard & J.
M. Wallace, John Wiley & Sons, Inc., (2000); An Introduction to Turbulent
Flow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

[0041] As used herein, the term "gas chromatography" or "GC" refers to
chromatography in which the sample mixture is vaporized and injected into
a stream of carrier gas (as nitrogen or helium) moving through a column
containing a stationary phase composed of a liquid or a particulate solid
and is separated into its component compounds according to the affinity
of the compounds for the stationary phase.

[0042] As used herein, the term "large particle column" or "extraction
column" refers to a chromatography column containing an average particle
diameter greater than about 50 μm.

[0043] As used herein, the term "analytical column" refers to a
chromatography column having sufficient chromatographic plates to effect
a separation of materials in a sample that elute from the column
sufficient to allow a determination of the presence or amount of an
analyte. In a preferred embodiment the analytical column contains
particles of about 5 μm in diameter. Such columns are often
distinguished from "extraction columns", which have the general purpose
of separating or extracting retained material from non-retained materials
in order to obtain a purified sample for further analysis.

[0044] As used herein, the terms "on-line" and "inline", for example as
used in "on-line automated fashion" or "on-line extraction" refers to a
procedure performed without the need for operator intervention. In
contrast, the term "off-line" as used herein refers to a procedure
requiring manual intervention of an operator. Thus, if samples are
subjected to precipitation, and the supernatants are then manually loaded
into an autosampler, the precipitation and loading steps are off-line
from the subsequent steps. In various embodiments of the methods, one or
more steps may be performed in an on-line automated fashion.

[0045] As used herein, the term "mass spectrometry" or "MS" refers to an
analytical technique to identify compounds by their mass. MS refers to
methods of filtering, detecting, and measuring ions based on their
mass-to-charge ratio, or "m/z". MS technology generally includes (1)
ionizing the compounds to form charged compounds; and (2) detecting the
molecular weight of the charged compounds and calculating a
mass-to-charge ratio. The compounds may be ionized and detected by any
suitable means. A "mass spectrometer" generally includes an ionizer and
an ion detector. In general, one or more molecules of interest are
ionized, and the ions are subsequently introduced into a mass
spectrometric instrument where, due to a combination of magnetic and
electric fields, the ions follow a path in space that is dependent upon
mass ("m") and charge ("z"). See, e.g., U.S. Pat. Nos. 6,204,500,
entitled "Mass Spectrometry From Surfaces;" 6,107,623, entitled "Methods
and Apparatus for Tandem Mass Spectrometry;" 6,268,144, entitled "DNA
Diagnostics Based On Mass Spectrometry;" 6,124,137, entitled
"Surface-Enhanced Photolabile Attachment And Release For Desorption And
Detection Of Analytes;" Wright et al., Prostate Cancer and Prostatic
Diseases 1999, 2: 264-76; and Merchant and Weinberger, Electrophoresis
2000, 21: 1164-67.

[0046] As used herein, the term "operating in negative ion mode" refers to
those mass spectrometry methods where negative ions are generated and
detected. The term "operating in positive ion mode" as used herein,
refers to those mass spectrometry methods where positive ions are
generated and detected.

[0047] As used herein, the term "ionization" or "ionizing" refers to the
process of generating an analyte ion having a net electrical charge equal
to one or more electron units. Negative ions are those having a net
negative charge of one or more electron units, while positive ions are
those having a net positive charge of one or more electron units.

[0048] As used herein, the term "electron ionization" or "EI" refers to
methods in which an analyte of interest in a gaseous or vapor phase
interacts with a flow of electrons. Impact of the electrons with the
analyte produces analyte ions, which may then be subjected to a mass
spectrometry technique.

[0049] As used herein, the term "chemical ionization" or "CI" refers to
methods in which a reagent gas (e.g. ammonia) is subjected to electron
impact, and analyte ions are formed by the interaction of reagent gas
ions and analyte molecules.

[0050] As used herein, the term "fast atom bombardment" or "FAB" refers to
methods in which a beam of high energy atoms (often Xe or Ar) impacts a
non-volatile sample, desorbing and ionizing molecules contained in the
sample. Test samples are dissolved in a viscous liquid matrix such as
glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,
2-nitrophenyloctyl ether, sulfolane, diethanolamine, and triethanolamine.
The choice of an appropriate matrix for a compound or sample is an
empirical process.

[0051] As used herein, the term "matrix-assisted laser desorption
ionization" or "MALDI" refers to methods in which a non-volatile sample
is exposed to laser irradiation, which desorbs and ionizes analytes in
the sample by various ionization pathways, including photo-ionization,
protonation, deprotonation, and cluster decay. For MALDI, the sample is
mixed with an energy-absorbing matrix, which facilitates desorption of
analyte molecules.

[0052] As used herein, the term "surface enhanced laser desorption
ionization" or "SELDI" refers to another method in which a non-volatile
sample is exposed to laser irradiation, which desorbs and ionizes
analytes in the sample by various ionization pathways, including
photo-ionization, protonation, deprotonation, and cluster decay. For
SELDI, the sample is typically bound to a surface that preferentially
retains one or more analytes of interest. As in MALDI, this process may
also employ an energy-absorbing material to facilitate ionization.

[0053] As used herein, the term "electrospray ionization" or "ESI," refers
to methods in which a solution is passed along a short length of
capillary tube, to the end of which is applied a high positive or
negative electric potential. Solution reaching the end of the tube is
vaporized (nebulized) into a jet or spray of very small droplets of
solution in solvent vapor. This mist of droplets flows through an
evaporation chamber, which is heated slightly to prevent condensation and
to evaporate solvent. As the droplets get smaller the electrical surface
charge density increases until such time that the natural repulsion
between like charges causes ions as well as neutral molecules to be
released.

[0054] As used herein, the term "atmospheric pressure chemical ionization"
or "APCI," refers to mass spectrometry methods that are similar to ESI;
however, APCI produces ions by ion-molecule reactions that occur within a
plasma at atmospheric pressure. The plasma is maintained by an electric
discharge between the spray capillary and a counter electrode. Then ions
are typically extracted into the mass analyzer by use of a set of
differentially pumped skimmer stages. A counterflow of dry and preheated
N2 gas may be used to improve removal of solvent. The gas-phase
ionization in APCI can be more effective than ESI for analyzing
less-polar species.

[0055] The term "atmospheric pressure photoionization" or "APPI" as used
herein refers to the form of mass spectrometry where the mechanism for
the photoionization of molecule M is photon absorption and electron
ejection to form the molecular ion M+. Because the photon energy
typically is just above the ionization potential, the molecular ion is
less susceptible to dissociation. In many cases it may be possible to
analyze samples without the need for chromatography, thus saving
significant time and expense. In the presence of water vapor or protic
solvents, the molecular ion can extract H to form MH+. This tends to
occur if M has a high proton affinity. This does not affect quantitation
accuracy because the sum of M+ and MH+ is constant. Drug compounds in
protic solvents are usually observed as MH+, whereas nonpolar compounds
such as naphthalene or testosterone usually form M+. See, e.g., Robb et
al., Anal. Chem. 2000, 72(15): 3653-3659.

[0056] As used herein, the term "inductively coupled plasma" or "ICP"
refers to methods in which a sample interacts with a partially ionized
gas at a sufficiently high temperature such that most elements are
atomized and ionized.

[0057] As used herein, the term "field desorption" refers to methods in
which a non-volatile test sample is placed on an ionization surface, and
an intense electric field is used to generate analyte ions.

[0058] As used herein, the term "desorption" refers to the removal of an
analyte from a surface and/or the entry of an analyte into a gaseous
phase. Laser diode thermal desorption (LDTD) is a technique wherein a
sample containing the analyte is thermally desorbed into the gas phase by
a laser pulse. The laser hits the back of a specially made 96-well plate
with a metal base. The laser pulse heats the base and the heat causes the
sample to transfer into the gas phase. The gas phase sample is then drawn
into an ionization source, where the gas phase sample is ionized in
preparation for analysis in the mass spectrometer. When using LDTD,
ionization of the gas phase sample may be accomplished by any suitable
technique known in the art, such as by ionization with a corona discharge
(for example by APCI).

[0059] As used herein, the term "selective ion monitoring" is a detection
mode for a mass spectrometric instrument in which only ions within a
relatively narrow mass range, typically about one mass unit, are
detected.

[0060] As used herein, "multiple reaction mode," sometimes known as
"selected reaction monitoring," is a detection mode for a mass
spectrometric instrument in which a precursor ion and one or more
fragment ions are selectively detected.

[0061] As used herein, the term "lower limit of quantification", "lower
limit of quantitation" or "LLOQ" refers to the point where measurements
become quantitatively meaningful. The analyte response at this LOQ is
identifiable, discrete and reproducible with a relative standard
deviation (RSD %) of less than 20% and an accuracy of 80% to 120%.

[0062] As used herein, the term "limit of detection" or "LOD" is the point
at which the measured value is larger than the uncertainty associated
with it. The LOD is the point at which a value is beyond the uncertainty
associated with its measurement and is defined as three times the RSD of
the mean at the zero concentration.

[0063] As used herein, an "amount" of an analyte in a body fluid sample
refers generally to an absolute value reflecting the mass of the analyte
detectable in volume of sample. However, an amount also contemplates a
relative amount in comparison to another analyte amount. For example, an
amount of an analyte in a sample can be an amount which is greater than a
control or normal level of the analyte normally present in the sample.

[0064] The term "about" as used herein in reference to quantitative
measurements not including the measurement of the mass of an ion, refers
to the indicated value plus or minus 10%. Mass spectrometry instruments
can vary slightly in determining the mass of a given analyte. The term
"about" in the context of the mass of an ion or the mass/charge ratio of
an ion refers to +/-0.50 atomic mass unit.

[0065] The summary of the invention described above is non-limiting and
other features and advantages of the invention will be apparent from the
following detailed description of the invention, and from the claims.

[0067] FIGS. 2A and 2B show exemplary calibration curves for 25OHD2
and 25OHD3 in serum samples determined by methods described in
Example 3.

[0068] FIG. 3A shows a plots of coefficient of variation versus
concentration for 25OHD2 and 25OHD3. FIG. 3B shows the same
plot expanded near the LLOQ. Details are described in Example 4.

[0069] FIGS. 4A-B show linear regression and Deming regression analyses
for the comparison of mass spectrometric determination of 25OHD2
with and without PTAD derivatization. Details are described in Example
10.

[0070] FIGS. 5A-B show linear regression and Deming regression analyses
for the comparison of mass spectrometric determination of 25OHD3
with and without PTAD derivatization. Details are described in Example
10.

[0071] FIGS. 6A-D show plots comparing the results of analysis of
multiplex samples and unmixed samples (with the same derivatization
agent). Details are described in Example 14.

[0072] FIGS. 7A-D are plots comparing the results of analysis of the same
specimen treated with different derivatization agents (but comparing
mixed versus mixed, or unmixed versus unmixed samples). Details are
described in Example 14.

[0073] FIGS. 8A-D are plots comparing the results of analysis of the same
specimen treated with different derivatization agents, with one analysis
coming from a mixed sample and one coming from an unmixed sample. Details
are described in Example 14.

[0074] FIG. 9A shows an exemplary Q1 scan spectrum (covering the m/z range
of about 350 to 450) for 25-hydroxyvitamin D2 ions. FIG. 9B shows an
exemplary product ion spectra (covering the m/z range of about 100 to
396) for fragmentation of the 25-hydroxyvitamin D2 precursor ion
with m/z of about 395.2. Details are described in Example 15.

[0075] FIG. 10A shows an exemplary Q1 scan spectrum (covering the m/z
range of about 350 to 450) for 25-hydroxyvitamin D3 ions. FIG. 10B
shows an exemplary product ion spectra (covering the m/z range of about
100 to 396) for fragmentation of the 25-hydroxyvitamin D3 precursor
ion with m/z of about 383.2. Details are described in Example 15.

[0076] FIG. 11A shows an exemplary Q1 scan spectrum (covering the m/z
range of about 520 to 620) for PTAD-25-hydroxyvitamin D2 ions. FIG.
11B shows an exemplary product ion spectra (covering the m/z range of
about 200 to 400) for fragmentation of the PTAD-25-hydroxyvitamin D2
precursor ion with m/z of about 570.3. Details are described in Example
15.

[0077] FIG. 12A shows an exemplary Q1 scan spectrum (covering the m/z
range of about 520 to 620) for PTAD-25-hydroxyvitamin D3 ions. FIG.
12B shows an exemplary product ion spectra (covering the m/z range of
about 200 to 400) for fragmentation of the PTAD-25-hydroxyvitamin D3
precursor ion with m/z of about 558.3. Details are described in Example
15.

[0078] FIG. 13A shows an exemplary Q1 scan spectrum (covering the m/z
range of about 520 to 620) for PTAD-1α,25-dihydroxyvitamin D2
ions. FIG. 13B shows an exemplary product ion spectra (covering the m/z
range of about 250 to 350) for fragmentation of the
PTAD-1α,25-dihydroxyvitamin D2 precursor ion with m/z of about
550.4. FIG. 13C shows an exemplary product ion spectra (covering the m/z
range of about 250 to 350) for fragmentation of the
PTAD-1α,25-dihydroxyvitamin D2 precursor ion with m/z of about
568.4. FIG. 13D shows an exemplary product ion spectra (covering the m/z
range of about 250 to 350) for fragmentation of the
PTAD-1α,25-dihydroxyvitamin D2 precursor ion with m/z of about
586.4. Details are described in Example 16.

[0079] FIG. 14A shows an exemplary Q1 scan spectrum (covering the m/z
range of about 520 to 620) for PTAD-1α,25-hydroxyvitamin D3
ions. FIG. 14B shows an exemplary product ion spectra (covering the m/z
range of about 250 to 350) for fragmentation of the
PTAD-1α,25-dihydroxyvitamin D3-PTAD precursor ion with m/z of
about 538.4. FIG. 14C shows an exemplary product ion spectra (covering
the m/z range of about 250 to 350) for fragmentation of the
PTAD-1α,25-dihydroxyvitamin D3 precursor ion with m/z of about
556.4. FIG. 14D shows an exemplary product ion spectra (covering the m/z
range of about 250 to 350) for fragmentation of the
PTAD-1α,25-dihydroxyvitamin D3 precursor ion with m/z of about
574.4. Details are described in Example 16.

[0080] FIG. 15A shows an exemplary Q1 scan spectrum (covering the m/z
range of about 500 to 620) for PTAD-vitamin D2 ions. FIG. 15B shows
an exemplary product ion spectra (covering the m/z range of about 250 to
350) for fragmentation of the PTAD-vitamin D2 precursor ion with m/z
of about 572.2. Details are described in Example 17.

[0081] FIG. 16A shows an exemplary Q1 scan spectrum (covering the m/z
range of about 500 to 620) for PTAD-vitamin D3 ions. FIG. 16B shows
an exemplary product ion spectra (covering the m/z range of about 250 to
350) for fragmentation of the PTAD-vitamin D3 precursor ion with m/z
of about 560.2. Details are described in Example 17.

DETAILED DESCRIPTION OF THE INVENTION

[0082] Methods are described for measuring steroidal compounds, such as
vitamin D and vitamin D related compounds, in a sample. More
specifically, methods are described for detecting and quantifying
steroidal compounds in a plurality of test samples in a single mass
spectrometric assay. The methods may utilize Cookson-type reagents, such
as PTAD, to generate derivatized steroidal compounds combined with
methods of mass spectrometry (MS), thereby providing a high-throughput
assay system for detecting and quantifying steroidal compounds in a
plurality of test samples. The preferred embodiments are particularly
well suited for application in large clinical laboratories for automated
steroidal compound quantification.

[0083] Suitable test samples for use in methods of the present invention
include any test sample that may contain the analyte of interest. In some
preferred embodiments, a sample is a biological sample; that is, a sample
obtained from any biological source, such as an animal, a cell culture,
an organ culture, etc. In certain preferred embodiments, samples are
obtained from a mammalian animal, such as a dog, cat, horse, etc.
Particularly preferred mammalian animals are primates, most preferably
male or female humans. Preferred samples comprise bodily fluids such as
blood, plasma, serum, saliva, cerebrospinal fluid, or tissue samples;
preferably plasma (including EDTA and heparin plasma) and serum; most
preferably serum. Such samples may be obtained, for example, from a
patient; that is, a living person, male or female, presenting oneself in
a clinical setting for diagnosis, prognosis, or treatment of a disease or
condition.

[0084] The present invention also contemplates kits for quantitation of
one or more steroidal compounds. A kit for a steroidal compound
quantitation assay may include a kit comprising the compositions provided
herein. For example, a kit may include packaging material and measured
amounts of an isotopically labeled internal standard, in amounts
sufficient for at least one assay. Typically, the kits will also include
instructions recorded in a tangible form (e.g., contained on paper or an
electronic medium) for using the packaged reagents for use in a steroidal
compound quantitation assay.

[0085] Calibration and QC pools for use in embodiments of the present
invention are preferably prepared using a matrix similar to the intended
sample matrix.

Sample Preparation for Mass Spectrometric Analysis

[0086] In preparation for mass spectrometric analysis, one or more
steroidal compounds may be enriched relative to one or more other
components in the sample (e.g. protein) by various methods known in the
art, including for example, liquid chromatography, filtration,
centrifugation, thin layer chromatography (TLC), electrophoresis
including capillary electrophoresis, affinity separations including
immunoaffinity separations, extraction methods including ethyl acetate or
methanol extraction, and the use of chaotropic agents or any combination
of the above or the like. These enrichment steps may be applied to
individual test samples prior to processing, individual processed samples
after derivatization, or to a multiplex sample after processed samples
have been combined.

[0087] Protein precipitation is one method of preparing a sample,
especially a biological sample, such as serum or plasma. Protein
purification methods are well known in the art, for example, Polson et
al., Journal of Chromatography B 2003, 785:263-275, describes protein
precipitation techniques suitable for use in methods of the present
invention. Protein precipitation may be used to remove most of the
protein from the sample leaving one or more steroidal compounds in the
supernatant. The samples may be centrifuged to separate the liquid
supernatant from the precipitated proteins; alternatively the samples may
be filtered to remove precipitated proteins. The resultant supernatant or
filtrate may then be applied directly to mass spectrometry analysis; or
alternatively to liquid chromatography and subsequent mass spectrometry
analysis. In certain embodiments, individual test samples, such as plasma
or serum, may be purified by a hybrid protein precipitation/liquid-liquid
extraction method. In these embodiments, an unprocessed test sample is
mixed with methanol, ethyl acetate, and water, and the resulting mixture
is vortexed and centrifuged. The resulting supernatant, containing one or
more purified steroidal compounds, is removed, dried to completion and
reconstituted in acetonitrile. The one or more purified steroidal
compounds in the acetonitrile solution may then be derivatized with any
Cookson-type reagent, preferably PTAD or an isotopically labeled variant
thereof.

[0088] Another method of sample purification that may be used prior to
mass spectrometry is liquid chromatography (LC). Certain methods of
liquid chromatography, including HPLC, rely on relatively slow, laminar
flow technology. Traditional HPLC analysis relies on column packing in
which laminar flow of the sample through the column is the basis for
separation of the analyte of interest from the sample. The skilled
artisan will understand that separation in such columns is a diffusional
process and may select LC, including HPLC, instruments and columns that
are suitable for use with derivatized steroidal compounds. The
chromatographic column typically includes a medium (i.e., a packing
material) to facilitate separation of chemical moieties (i.e.,
fractionation). The medium may include minute particles, or may include a
monolithic material with porous channels. A surface of the medium
typically includes a bonded surface that interacts with the various
chemical moieties to facilitate separation of the chemical moieties. One
suitable bonded surface is a hydrophobic bonded surface such as an alkyl
bonded, cyano bonded surface, or highly pure silica surface. Alkyl bonded
surfaces may include C-4, C-8, C-12, or C-18 bonded alkyl groups. In
preferred embodiments, the column is a highly pure silica column (such as
a Thermo Hypersil Gold Aq column). The chromatographic column includes an
inlet port for receiving a sample and an outlet port for discharging an
effluent that includes the fractionated sample. The sample may be
supplied to the inlet port directly, or from an extraction column, such
as an on-line SPE cartridge or a TFLC extraction column. In preferred
embodiments, a multiplex sample may be purified by liquid chromatography
prior to mass spectrometry.

[0089] In one embodiment, the multiplex sample may be applied to the LC
column at the inlet port, eluted with a solvent or solvent mixture, and
discharged at the outlet port. Different solvent modes may be selected
for eluting the analyte(s) of interest. For example, liquid
chromatography may be performed using a gradient mode, an isocratic mode,
or a polytyptic (i.e. mixed) mode. During chromatography, the separation
of materials is effected by variables such as choice of eluent (also
known as a "mobile phase"), elution mode, gradient conditions,
temperature, etc.

[0090] In certain embodiments, analytes may be purified by applying a
multiplex sample to a column under conditions where analytes of interest
are reversibly retained by the column packing material, while one or more
other materials are not retained. In these embodiments, a first mobile
phase condition can be employed where the analytes of interest are
retained by the column, and a second mobile phase condition can
subsequently be employed to remove retained material from the column once
the non-retained materials are washed through. Alternatively, analytes
may be purified by applying a multiplex sample to a column under mobile
phase conditions where the analytes of interest elute at a differential
rates in comparison to one or more other materials. Such procedures may
enrich the amount of an analyte of interest in the eluent at a particular
time (i.e., a characteristic retention time) relative to one or more
other components of the sample.

[0091] In one preferred embodiment, HPLC is conducted with an alkyl bonded
analytical column chromatographic system. In certain preferred
embodiments, a highly pure silica column (such as a Thermo Hypersil Gold
Aq column) is used. In certain preferred embodiments, HPLC and/or TFLC
are performed using HPLC Grade water as mobile phase A and HPLC Grade
ethanol as mobile phase B.

[0092] By careful selection of valves and connector plumbing, two or more
chromatography columns may be connected as needed such that material is
passed from one to the next without the need for any manual steps. In
preferred embodiments, the selection of valves and plumbing is controlled
by a computer pre-programmed to perform the necessary steps. Most
preferably, the chromatography system is also connected in such an
on-line fashion to the detector system, e.g., an MS system. Thus, an
operator may place a tray of samples in an autosampler, and the remaining
operations are performed under computer control, resulting in
purification and analysis of all samples selected.

[0093] In some embodiments, an extraction column may be used for
purification of steroidal compounds prior to mass spectrometry. In such
embodiments, samples may be extracted using a extraction column which
captures the analyte, then eluted and chromatographed on a second
extraction column or on an analytical HPLC column prior to ionization.
For example, sample extraction with a TFLC extraction column may be
accomplished with a large particle size (50 μm) packed column. Sample
eluted off of this column may then be transferred to an HPLC analytical
column for further purification prior to mass spectrometry. Because the
steps involved in these chromatography procedures may be linked in an
automated fashion, the requirement for operator involvement during the
purification of the analyte can be minimized. This feature may result in
savings of time and costs, and eliminate the opportunity for operator
error.

[0094] In some embodiments, protein precipitation is accomplished with a
hybrid protein precipitation/liquid-liquid extraction method which
includes methanol protein precipitation and ethyl acetate/water
extraction from serum test samples. The resulting steroidal compounds may
be derivatized prior to being subjected to an extraction column.
Preferably, the hybrid protein precipitation/liquid-liquid extraction
method and the extraction column are connected in an on-line fashion. In
preferred embodiments where the steroidal compounds are selected from the
group consisting of vitamin D and vitamin D related compounds, the
extraction column is preferably a C-8 extraction column, such as a
Cohesive Technologies C8XL online extraction column (50 μm particle
size, 0.5×50 mm) or equivalent. The eluent from the extraction
column may then be applied to an analytical LC column, such as a HPLC
column in an on-line fashion, prior to mass spectrometric analysis.
Because the steps involved in these chromatography procedures may be
linked in an automated fashion, the requirement for operator involvement
during the purification of the analyte can be minimized. This feature may
result in savings of time and costs, and eliminate the opportunity for
operator error.

Detection and Quantitation by Mass Spectrometry

[0095] In various embodiments, derivatized steroidal compounds may be
ionized by any method known to the skilled artisan. Mass spectrometry is
performed using a mass spectrometer, which includes an ion source for
ionizing the fractionated sample and creating charged molecules for
further analysis. For example ionization of the sample may be performed
by electron ionization, chemical ionization, electrospray ionization
(ESI), photon ionization, atmospheric pressure chemical ionization
(APCI), photoionization, atmospheric pressure photoionization (APPI),
fast atom bombardment (FAB), liquid secondary ionization (LSI), matrix
assisted laser desorption ionization (MALDI), field ionization, field
desorption, thermospray/plasmaspray ionization, surface enhanced laser
desorption ionization (SELDI), inductively coupled plasma (ICP), particle
beam ionization, and LDTD. The skilled artisan will understand that the
choice of ionization method may be determined based on the analyte to be
measured, type of sample, the type of detector, the choice of positive
versus negative mode, etc.

[0096] Derivatized steroidal compounds may be ionized in positive or
negative mode. In preferred embodiments, derivatized steroidal compounds
are ionized by APCI in positive mode. In related preferred embodiments,
derivatized steroidal compounds ions are in a gaseous state and the inert
collision gas is argon or nitrogen; preferably argon.

[0097] In mass spectrometry techniques generally, after the sample has
been ionized, the positively or negatively charged ions thereby created
may be analyzed to determine a mass-to-charge ratio. Suitable analyzers
for determining mass-to-charge ratios include quadrupole analyzers, ion
traps analyzers, and time-of-flight analyzers. Exemplary ion trap methods
are described in Bartolucci, et al., Rapid Commun. Mass Spectrom. 2000,
14:967-73.

[0098] The ions may be detected using several detection modes. For
example, selected ions may be detected, i.e. using a selective ion
monitoring mode (SIM), or alternatively, mass transitions resulting from
collision induced dissociation or neutral loss may be monitored, e.g.,
multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).
Preferably, the mass-to-charge ratio is determined using a quadrupole
analyzer. For example, in a "quadrupole" or "quadrupole ion trap"
instrument, ions in an oscillating radio frequency field experience a
force proportional to the DC potential applied between electrodes, the
amplitude of the RF signal, and the mass/charge ratio. The voltage and
amplitude may be selected so that only ions having a particular
mass/charge ratio travel the length of the quadrupole, while all other
ions are deflected. Thus, quadrupole instruments may act as both a "mass
filter" and as a "mass detector" for the ions injected into the
instrument.

[0099] One may enhance the resolution of the MS technique by employing
"tandem mass spectrometry," or "MS/MS". In this technique, a precursor
ion (also called a parent ion) generated from a molecule of interest can
be filtered in an MS instrument, and the precursor ion subsequently
fragmented to yield one or more fragment ions (also called daughter ions
or product ions) that are then analyzed in a second MS procedure. By
careful selection of precursor ions, only ions produced by certain
analytes are passed to the fragmentation chamber, where collisions with
atoms of an inert gas produce the fragment ions. Because both the
precursor and fragment ions are produced in a reproducible fashion under
a given set of ionization/fragmentation conditions, the MS/MS technique
may provide an extremely powerful analytical tool. For example, the
combination of filtration/fragmentation may be used to eliminate
interfering substances, and may be particularly useful in complex
samples, such as biological samples.

[0101] The results of an analyte assay may be related to the amount of the
analyte in the original sample by numerous methods known in the art. For
example, given that sampling and analysis parameters are carefully
controlled, the relative abundance of a given ion may be compared to a
table that converts that relative abundance to an absolute amount of the
original molecule. Alternatively, external standards may be run with the
samples, and a standard curve constructed based on ions generated from
those standards. Using such a standard curve, the relative abundance of a
given ion may be converted into an absolute amount of the original
molecule. In certain preferred embodiments, an internal standard is used
to generate a standard curve for calculating the quantity of steroidal
compounds. Methods of generating and using such standard curves are well
known in the art and one of ordinary skill is capable of selecting an
appropriate internal standard. For example, in some embodiments, one or
more isotopically labeled vitamin D metabolites (e.g., 25OHD2-[6,
19, 19]-2H3 and 25OHD3-[6, 19, 19]-2H3) may be
used as internal standards. Numerous other methods for relating the
amount of an ion to the amount of the original molecule will be well
known to those of ordinary skill in the art.

[0102] One or more steps of the methods may be performed using automated
machines. In certain embodiments, one or more purification steps are
performed on-line, and more preferably all of the purification and mass
spectrometry steps may be performed in an on-line fashion.

[0103] In certain mass spectrometry techniques, such as MS/MS, precursor
ions are isolated for further fragmentation though collision activated
dissociation (CAD). In CAD, precursor ions gain energy through collisions
with an inert gas, and subsequently fragment by a process referred to as
"unimolecular decomposition." Sufficient energy must be deposited in the
precursor ion so that certain bonds within the ion can be broken due to
increased vibrational energy.

[0104] Steroidal compounds in a sample may be detected and/or quantified
using MS/MS as follows. The samples may first be purified by protein
precipitation or a hybrid protein precipitation/liquid-liquid extraction.
Then, one or more steroidal compounds in the purified sample are
derivatized with a Cookson-type reagent, such as PTAD or an isotopic
variant thereof. The purified samples may then subjected to liquid
chromatography, preferably on an extraction column (such as a TFLC
column) followed by an analytical column (such as a HPLC column); the
flow of liquid solvent from a chromatographic column enters the nebulizer
interface of an MS/MS analyzer; and the solvent/analyte mixture is
converted to vapor in the heated charged tubing of the interface. The
analyte(s) (e.g., derivatized steroidal compounds such as derivatized
vitamin D metabolites), contained in the solvent, are ionized by applying
a large voltage to the solvent/analyte mixture. As the analytes exit the
charged tubing of the interface, the solvent/analyte mixture nebulizes
and the solvent evaporates, leaving analyte ions. Alternatively,
derivatized steroidal compounds in the purified samples may not be
subject to liquid chromatography prior to ionization. Rather, the samples
may be spotted in a 96-well plate and volatilized and ionized via LDTD.

[0105] The ions, e.g. precursor ions, pass through the orifice of a tandem
mass spectrometric (MS/MS) instrument and enter the first quadrupole. In
a tandem mass spectrometric instrument, quadrupoles 1 and 3 (Q1 and Q3)
are mass filters, allowing selection of ions (i.e., selection of
"precursor" and "fragment" ions in Q1 and Q3, respectively) based on
their mass to charge ratio (m/z). Quadrupole 2 (Q2) is the collision
cell, where ions are fragmented. The first quadrupole of the mass
spectrometer (Q1) selects for molecules with the mass to charge (m/z)
ratios of derivatized steroidal compounds of interest. Precursor ions
with the correct mass/charge ratios are allowed to pass into the
collision chamber (Q2), while unwanted ions with any other mass/charge
ratio collide with the sides of the quadrupole and are eliminated.
Precursor ions entering Q2 collide with neutral argon gas molecules and
fragment. The fragment ions generated are passed into quadrupole 3 (Q3),
where the fragment ions of derivatized steroidal compounds of interest
are selected while other ions are eliminated.

[0106] The methods may involve MS/MS performed in either positive or
negative ion mode; preferably positive ion mode. Using standard methods
well known in the art, one of ordinary skill is capable of identifying
one or more fragment ions of a particular precursor ion of derivatized
steroidal compounds that may be used for selection in quadrupole 3 (Q3).

[0107] As ions collide with the detector they produce a pulse of electrons
that are converted to a digital signal. The acquired data is relayed to a
computer, which plots counts of the ions collected versus time. The
resulting mass chromatograms are similar to chromatograms generated in
traditional HPLC-MS methods. The areas under the peaks corresponding to
particular ions, or the amplitude of such peaks, may be measured and
correlated to the amount of the analyte of interest. In certain
embodiments, the area under the curves, or amplitude of the peaks, for
fragment ion(s) and/or precursor ions are measured to determine the
amount of a particular steroidal compounds. As described above, the
relative abundance of a given ion may be converted into an absolute
amount of the original analyte using calibration standard curves based on
peaks of one or more ions of an internal molecular standard.

Processing Patient Samples for Analysis of Multiplex Patient Samples

[0108] Following the procedures outlined above, multiple patient samples
can be multiplex (i.e., mixed and assayed together) if each patient
sample is processed differently. The phrase "processed differently" means
that each patient sample to be included in the multiplex sample is
processed in such a way that steroidal compounds in two or more patient
samples that are originally indistinguishable by mass spectrometry become
distinguishable after processing. This may be accomplished by processing
each patient sample with a different agent that derivitizes steroidal
compounds. The derivatizing agents selected for use must generate
derivatized steroidal compounds that are distinguishable by mass
spectrometry. The basis for distinguishing derivatized steroidal
compounds by mass spectrometry will be a difference in the mass of ions
from the derivatized steroidal compounds. The differences in mass may
arise from the use of two or more different derivatizing agents, such as
PTAD and DMEQTAD. Differences in mass may also arise from the use of two
or more isotopic variants of the same derivatizing agent, such as PTAD
and 13C6-PTAD. These two approaches are not mutually exclusive,
and any combination of different derivatizing agents and isotopic
variants of the same agent may be used to uniquely label steroidal
compounds in each patient sample in the plurality of patient samples to
be analyzed. Optionally, one sample from the plurality of patient samples
may be processed without a derivatizing agent.

[0109] After processing a plurality of patient samples, a particular
steroidal compound from one patient sample will have a different mass
spectrometric profile than the same steroidal compound in other patient
samples. When processed patient samples are mixed to form a multiplex
sample which is then analyzed to determine the levels of processed
steroidal compounds, the differences in mass spectrometric profiles of
the detected processed steroidal compounds allow for each processed
steroidal compound to be attributed to an originating patient sample.
Thus, the amounts of a steroidal compound in two or more patient samples
are determined by a single mass spectrometric analysis of a multiplex
sample.

[0110] As indicated above, different Cookson-type reagents may be used as
derivatizing agents for different patient samples; for example, one
patient sample may be derivatized with PTAD, and a second patient sample
derivatized with DMEQTAD. Using different Cookson-type reagents generally
results in large mass differences between the derivatized analytes. For
example, the difference in mass between a steroidal compound derivatized
with PTAD and the same compound derivatized with DMEQTAD is about 200
mass units (the mass difference between PTAD and DMEQTAD).

[0111] Isotopic variants of the same Cookson-type reagent may also be used
to create distinguishable derivatives in multiple patient samples. For
example, one patient sample may be derivatized with PTAD, and a second
patient sample may be derivatized with 13C6-PTAD. In this
example, the difference in mass between PTAD and 13C6-PTAD is
about 6 mass units.

[0112] The following Examples serve to illustrate the invention through
processing multiple patient samples with isotopic variants of PTAD. These
Examples are in no way intended to limit the scope of the methods. In
particular, the following Examples demonstrate quantitation of vitamin D
metabolites by mass spectrometry with the use of 25OHD2-[6, 19,
19]-2H3 or 25OHD3-[6, 19, 19]-2H3 as internal
standards. Demonstration of the methods of the present invention as
applied to vitamin D metabolites does not limit the applicability of the
methods to only vitamin D and vitamin D related compounds. Similarly, the
use of 25OHD2-[6, 19, 19]-2H3 or 25OHD3-[6, 19,
19]-2H3 as internal standards are not meant to be limiting in
any way. Any appropriate chemical species, easily determined by one in
the art, may be used as an internal standard for steroidal compound
quantitation.

[0113] The following automated hybrid protein precipitation/liquid-liquid
extraction technique was conducted on patient serum samples. Gel Barrier
Serum (i.e., serum collected in Serum Separator Tubes) as well as EDTA
plasma and Heparin Plasma have also been established as acceptable for
this assay.

[0114] A Perkin-Elmer Janus robot and a TomTec Quadra Tower robot was used
to automate the following procedure. For each sample, 50 μL of serum
was added to a well of a 96 well plate. Then 25 μL of internal
standard cocktail (containing isotopically labeled 25OHD2-[6, 19,
19]-2H3 and 25OHD3-[6, 19, 19]-2H3) was added to
each well, and the plate vortexed. Then 75 μL of methanol was added,
followed by additional vortexing. 300 μL of ethyl acetate and 75 μL
of water was then added, followed by additional vortexing,
centrifugation, and transfer of the resulting supernatant to a new
96-well plate.

[0115] The transferred liquid in the second 96-well plate from Example 1
was dried to completion under a flowing nitrogen gas manifold.
Derivatization was accomplished by adding 100 μL of a 0.1 mg/mL
solution of the Cookson-type derivatization agent PTAD in acetonitrile to
each well. The derivatization reaction was allowed to proceed for
approximately one hour, and was quenched by adding 100 μL of water to
the reaction mixture.

Example 2

Extraction of Vitamin D Metabolites with Liquid Chromatography

[0116] Sample injection was performed with a Cohesive Technologies Aria
TX-4 TFLC system using Aria OS V 1.5.1 or newer software.

[0117] The TFLC system automatically injected an aliquot of the above
prepared samples into a Cohesive Technologies C8XL online extraction
column (50 μm particle size, 005×50 mm, from Cohesive
Technologies, Inc.) packed with large particles. The samples were loaded
at a high flow rate to create turbulence inside the extraction column.
This turbulence ensured optimized binding of derivatized vitamin D
metabolites to the large particles in the column and the passage of
excess derivatizing reagent and debris to waste.

[0118] Following loading, the sample was eluted off to the analytical
column, a Thermo Hypersil Gold Aq analytical column (5 μm particle
size, 50×2.1 mm), with a water/ethanol elution gradient. The HPLC
gradient was applied to the analytical column, to separate vitamin D
metabolites from other analytes contained in the sample. Mobile phase A
was water and mobile phase B was ethanol. The HPLC gradient started with
a 35% organic gradient which was ramped to 99% in approximately 65
seconds.

Example 3

Detection and Quantitation of Derivatized Vitamin D Metabolites by MS/MS

[0119] MS/MS was performed using a Finnigan TSQ Quantum Ultra MS/MS system
(Thermo Electron Corporation). The following software programs, all from
Thermo Electron, were used in the Examples described herein: Quantum Tune
Master V 1.5 or newer, Xcalibur V 2.07 or newer, LCQuan V 2.56 (Thermo
Finnigan) or newer, and ARIA OS v1.5.1 (Cohesive Technologies) or newer.
Liquid solvent/analyte exiting the analytical column flowed to the
nebulizer interface of the MS/MS analyzer. The solvent/analyte mixture
was converted to vapor in the tubing of the interface. Analytes in the
nebulized solvent were ionized by ESI.

[0120] Ions passed to the first quadrupole (Q1), which selected ions for a
derivatized vitamin D metabolite. Ions with a m/z of 570.32±0.50 were
selected for PTAD-25OHD2; ions with a m/z of 558.32±0.50 were
selected for PTAD-25OHD3. Ions entering quadrupole 2 (Q2) collided
with argon gas to generate ion fragments, which were passed to quadrupole
3 (Q3) for further selection. Mass spectrometer settings are shown in
Table 1. Simultaneously, the same process using isotope dilution mass
spectrometry was carried out with internal standards,
PTAD-25OHD2-[6, 19, 19]-2H3 and PTAD-25OHD3-[6, 19,
19]-2H3. The following mass transitions were used for detection
and quantitation during validation on positive polarity. The indicated
mass transitions are not meant to be limiting in any way. As seen in the
Examples that follow, other mass transitions could be selected for each
analyte to generate quantitative data.

[0123] The LLOQ is the point where measurements become quantitatively
meaningful. The analyte response at this LLOQ is identifiable, discrete
and reproducible with a precision (i.e., coefficient of variation (CV))
of greater than 20% and an accuracy of 80% to 120%. The LLOQ was
determined by assaying five different human serum samples spiked with
PTAD-25OHD2 and PTAD-25OHD3 at levels near the expected LLOQ
and evaluating the reproducibility. Analysis of the collected data
indicates that samples with concentrations of about 4 ng/mL yielded CVs
of about 20%. Thus, the LLOQ of this assay for both PTAD-25OHD2 and
PTAD-25OHD3 was determined to be about 4 ng/mL. The graphical
representations of CV versus concentration for both analytes are shown in
FIGS. 3A-B (FIG. 3A shows the plots over an expanded concentration range,
while FIG. 3B shows the same plot expanded near the LOQ).

[0124] The LOD is the point at which a value is beyond the uncertainty
associated with its measurement and is defined as three standard
deviations from the zero concentration. To determine the LOD, generally,
blank samples of the appropriate matrix are obtained and tested for
interferences. However, no appropriate biological matrix could be
obtained where the endogenous concentration of 25OHD3 is zero, so a
solution of 5% bovine serum albumin in phosphate buffered saline (with an
estimated 1.5 ng/mL 25OHD3) was used for LOD studies. The standard
was run in 20 replicates each and the resulting area rations were
statistically analyzed to determine that the LOD for 25OHD2 and
25OHD3 are about 1.9 and 3.3 ng/mL, respectively. Raw data from
these studies is presented in Table 3, below

[0125] Linearity of derivatized vitamin D metabolite detection in the
assay was determined by diluting four pools serum with high endogenous
concentration of either 25OHD2 or 25OHD3 and analyzing
undiluted specimens and diluted specimens at 1:2, 1:4, and 1:8, in
quadruplicate. Quadratic regression of the data was performed yielding
correlation coefficients across the concentration range tested of
R2=0.97. These studies demonstrated that specimens may be diluted at
1:4 with average recovery of 101%, permitting a reportable range of about
4 to about 512 ng/mL. Average measured values for each of the specimen
dilution levels and correlation values from linear regression analysis
are presented in Table 4A, below. Percent recoveries for each of the
specimen dilution levels are presented in Table 4B, below.

[0126] The specificity of the assay against similar analytes was
determined to have no cross reactivity for any vitamin D metabolite
tested with the exception of 3-epi-25OHD3, which behaves similarly
to 25OHD3 in the assay. The side-chain labeled stable isotopes of
25OHD2 and 25OHD3 also showed cross-reactivity owing to hydrogen
exchange that occurs in the ion source. Thus, side-chain labeled stable
isotopes of 25OHD2 and 25OHD3 should not be used as internal
standards. Table 5, below, shows the compounds tested and the results of
the cross-reactivity studies.

[0127] Six standards at 5, 15, 30, 60, 90, and 120 ng/mL for each analyte
were run in every assay as a means as quantitating reproducibility. The
day-to-day reproducibility was determined using calibration curves from
19 assays. The data from these 19 assays are presented in Tables 6A (for
25OHD2) and 6B (for 25OHD3).

[0128] Intra-assay variation is defined as the reproducibility of results
for a sample within a single assay. To assess intra-assay variation,
twenty replicates from each of four quality control (QC) pools covering
the reportable range of the assay were prepared and measured from pooled
serum with 25OHD2 and 25OHD3 at arbitrary ultralow, low,
medium, and high concentrations for each analyte. Acceptable levels for
the coefficient of variation (CV) are less then 15% for the three higher
concentration, and less than 20% for the lowest concentration (at or near
the LOQ for the assay).

[0129] The results of the intra-assay variation studies indicate that the
CV for the four QC pools are 9.1%, 6.4%, 5.0%, and 5.9% with mean
concentrations of 13.7 ng/mL, 30.0 ng/mL, 52.4 ng/mL, and 106.9 ng/mL,
respectively, for PTAD-25OHD2, and 3.5%, 4.9%, 5.1%, and 3.3% with
mean concentrations of 32.8 ng/mL, 15.0 ng/mL, 75.4 ng/mL, and 102.3
ng/mL, respectively, for PTAD-25OHD3. The data from analysis of
these replicates is shown in Tables 7A and 7B.

[0130] Five aliquots of each of the same four QC pools were assayed over
six days to determine the coefficient of variation (CV) between assays.
The results of the intra-assay variation studies indicate that the
inter-assay CV for the four QC pools are about 8.3%, 6.2%, 8.1%, and 6.4%
with mean concentrations of about 13.1 ng/mL, 29.8 ng/mL, 51.9 ng/mL, and
107.8 ng/mL, respectively, for PTAD-25OHD2, and about 4.8%, 6.7%,
4.7%, and 6.7% with mean concentrations of about 31.1 ng/mL, 14.5 ng/mL,
75.1 ng/mL, and 108.4 ng/mL, respectively, for PTAD-25OHD3. The data
from analysis of these replicates is shown in Tables 8A and 8B.

[0131] Two recovery studies were performed. The first was performed using
six specimens, spiked with two different concentrations each of
25OHD2 and 25OHD3. These spiked specimens were subjected to the
hybrid protein precipitation/liquid-liquid extraction procedure described
in Example 1. Then, aliquots of the extracts of the spiked specimens were
derivatized with normal PTAD, following the procedure discussed above,
and analyzed in quadruplicate. The spiked concentrations were within the
workable range of the assay. The six pools yielded an average accuracy of
about 89% at spiked levels of greater than about 44 ng/mL and about 92%
at spiked levels of greater than about 73 ng/mL. Only two of the 24
experimental recoveries were less than 85%; the remaining 22 assays were
within the acceptable accuracy range of 85-115%. The results of the
spiked specimen recovery studies are presented in Table 9, below.

[0132] The second recovery study was performed again using six specimens.
Of these six specimens, three had high endogenous concentration of
25OHD2 and three had high endogenous concentrations of 25OHD3.
The specimens were paired and mixed at ratios of about 4:1, 1:1, and 1:4.
The resulting mixtures were subjected to the hybrid protein
precipitation/liquid-liquid extraction procedure described in Example 1.
Then, aliquots of the extracts of the mixed specimens were derivatized
with normal PTAD, following the procedure discussed above, and analyzed
in quadruplicate. These experiments yielded an average accuracy of about
98% for 25OHD2 and about 93% for 25OHD3. All individual results
were within the acceptable accuracy range of 85-115%. The results of the
mixed specimen recovery studies are presented in Table 10, below.

[0133] The method of detecting vitamin D metabolites following
PTAD-derivatization was compared to a mass spectrometric method in which
the vitamin D metabolites are not derivatized prior to analysis. Such a
method is described in the published U.S. Patent Application 2006/0228808
(Caulfield, et al.). Eight specimens were split and analyzed according to
both methods. The correlation between the two methods was assessed with
linear regression, deming regression, and Bland-Altman analysis for
complete data sets (including calibration samples, QC pools, and
unknowns), as well as for unknowns only.

[0135] The effect hemolysis, lipemia, and icteria have on the assay was
also investigated.

[0136] Hemolysis.

[0137] The effect of hemolysis was evaluated by pooling patient samples
with known endogenous concentrations of both 25OHD2 and 25OHD3
to create five different pools with concentrations across the dynamic
range of the assay. Then, lysed whole blood was spiked into the pools to
generate lightly and moderately hemolyzed samples.

[0138] The lightly and moderately hemolyzed samples were analyzed in
quadruplicate and the results were compared to levels of samples without
whole blood spikes. The resulting comparison indicated a % difference of
less than 15% for both 25OHD2 and 25OHD3. Therefore, light to
moderately hemolyzed specimens are acceptable for analysis.

[0139] Lipemia.

[0140] The effect of lipemia was evaluated by pooling patient samples with
known endogenous concentrations of both 25OHD2 and 25OHD3 to
create five different pools with concentrations across the dynamic range
of the assay. Then, powdered lipid extract was added to the pools to
generate lightly and grossly lipemic specimens. Specimens were run in
quadruplicate and results were compared to the non-lipemic pool result
and the accuracy was calculated. The data shows that determination of
25OHD2 is unaffected by lipemia (all values were within an
acceptable accuracy range of 85-115%), however, 25OHD3 is affected
by lipemia, resulting in determination in lower than expected values. The
degree of variance increased with the degree of lipemia. Therefore, light
but not grossly lipemic specimens are acceptable.

[0141] Icteria.

[0142] The effect of icteria was evaluated by pooling patient samples with
known endogenous concentrations of both 25OHD2 and 25OHD3 to
create five different pools with concentrations across the dynamic range
of the assay. Then, a concentrated solution of Bilirubin was spiked into
the pools to generate lightly and grossly icteric specimens. Specimens
were run in quadruplicate and results were compared to the non-icteric
pool result and the accuracy was calculated. The data showed that
25OHD2 and 25OHD3 are unaffected by icteria (with all values
within an acceptable accuracy range of 85-115%). Therefore, icteric
specimens are acceptable.

Example 12

Injector Carryover Studies

[0143] Blank matrices were run immediately after a specimen with a high
concentration of 25OHD2 and 25OHD3 in order to evaluate
carryover between samples. These studies indicated that the response at
the retention time of analyte or internal standard was not large enough
to compromise the integrity of the assay. Data from these studies is
presented in Table 11, below.

[0144] The assay was conducted on various specimen types. Human serum and
Gel-Barrier Serum (i.e., serum from Serum Separator Tubes), as well as
EDTA Plasma and Heparin were established as acceptable sample types. In
these studies, sets of human serum (serum), Gel-Barrier Serum (SST), EDTA
Plasma (EDTA), and heparin (Na Hep) drawn at the same time from the same
patient were tested for 25OHD2 (40 specimen sets) and 25OHD3 (6
specimen sets). Due to the limitations with clot detection/sensing in
existing automated pipetting systems, plasma was not tested for automated
procedures.

[0145] The results of the specimen type studies are presented in Tables
12A and B for 25OHD2 and 25OHD3, respectively.

[0146] Patient sample multiplexing after derivatization with different
derivatizing agents was demonstrated in the following crossover
experiments.

[0147] First, two patients samples (i.e., sample A and sample B) were both
subjected to the hybrid protein precipitation/liquid-liquid extraction
procedure described in Example 1. Then, aliquots of the extracts from
sample A and sample B were derivatized with normal PTAD, following the
procedure discussed above. Second aliquots of the extracts from sample A
and sample B were also derivatized with 13C6-PTAD, also
according to the procedure discussed above.

[0148] After the four derivatization reactions were quenched, a portion of
the PTAD-derivatized sample A was mixed with
13C6-PTAD-derivatized sample B, and a portion of
13C6-PTAD-derivatized sample A was mixed with PTAD-derivatized
sample B.

[0149] These mixtures were loaded onto a 96-well plate and analyzed
according to the liquid chromatography-mass spectrometry methods
described in Examples 2 and 3. Again, 25OHD2-[6, 19,
19]-2H3 and 25OHD3-[6, 19, 19]-2H3 were used as
internal standards (shown in Table 13, below, as 25OHD2-IS and
25OHD3-IS). The mass spectrometer was programmed to monitor for the
PTAD- and 13C6-PTAD-derivatized vitamin D metabolite conjugates
shown in Table 13. The indicated mass transitions are not meant to be
limiting in any way. As seen in the Examples that follow, other mass
transitions could be selected for each analyte to generate quantitative
data.

[0150] Derivatized samples A and B and permutations of mixtures of the two
described above were analyzed and plotted to evaluate goodness of fit of
the data. These results are presented in FIGS. 6A-D, 7A-D, and 8A-D.

[0151] FIGS. 6A-D are plots comparing the results of analysis of multiplex
samples and unmixed samples (with the same derivatization agent). These
plots show R2 values in excellent agreement (i.e., R2 values
for all four variants are in excess of 0.98). This shows that, given a
constant derivatization agent, analysis of mixed samples gives the same
result as analysis of unmixed samples.

[0152] FIGS. 7A-D are plots comparing the results of analysis of the same
specimen treated with different derivatization agents (but comparing
mixed versus mixed, or unmixed versus unmixed samples). These plots also
show R2 values in excellent agreement (i.e., R2 values for all
four variants are in excess of 0.98). This shows that the isotopic
variation between PTAD and 13C6-PTAD is not a source of
difference in the performance of the assay, at least when the compared
samples are both mixed, or unmixed.

[0153] FIGS. 8A-D are plots comparing the results of analysis of the same
specimen treated with different derivatization agents, with one analysis
coming from a mixed sample and one coming from an unmixed sample. These
plots also show R2 values in excellent agreement (i.e., R2
values for all four variants are in excess of 0.99). This shows that the
isotopic variation between PTAD and 13C6-PTAD in combination
with variation between mixed and unmixed samples is not a source of
difference in the performance of the assay.

[0154] Thus, isotopic variation of the PTAD derivatization agent made no
meaningful difference even when samples were mixed together and
introduced into the mass spectrometer as a single injection. Multiplexing
of patient samples was successfully demonstrated.

[0155] Underivatized and PTAD derivatized 25-hydroxyvitamin D2 and
25-hydroxyvitamin D3 were analyzed by LDTD-MS/MS. Results of these
analyses are presented below.

[0156] Exemplary Q1 scan spectra from analysis of 25-hydroxyvitamin
D2 and 25-hydroxyvitamin D3 are shown in FIGS. 9A and 10A,
respectively. These spectra were collected by scanning Q1 across a m/z
range of about 350 to 450.

[0157] Exemplary product ion scans from each of these species are
presented in FIGS. 9B and 10B, respectively. The precursor ions selected
in Q1, and collision energies used in fragmenting the precursors are
indicated in Table 14.

[0158] A preferred MRM transition for the quantitation of
25-hydroxyvitamin D2 is fragmenting a precursor ion with a m/z of
about 395.2 to a product ion with a m/z of about 208.8 or 251.0. A
preferred MRM transition for the quantitation of 25-hydroxyvitamin
D3 is fragmenting a precursor ion with a m/z of about 383.2 to a
product ion with a m/z of about 186.9 or 257.0. However, as can be seen
in the product ion scans in FIGS. 9B and 10B, additional product ions may
be selected to replace or augment the preferred fragment ion.

[0159] Exemplary Q1 scan spectra from the analysis of samples containing
PTAD-25-hydroxyvitamin D2 and PTAD-25-hydroxyvitamin D3 are
shown in FIGS. 11A and 12A, respectively. These spectra were collected by
scanning Q1 across a m/z range of about 520 to 620.

[0160] Exemplary product ion scans from each of these species are
presented in FIGS. 11B and 12B, respectively. The precursor ions selected
in Q1, and collision energies used in fragmenting the precursors are
indicated in Table 15.

[0161] A preferred MRM transition for the quantitation of
PTAD-25-hydroxyvitamin D2 is fragmenting a precursor ion with a m/z
of about 570.3 to a product ion with a m/z of about 298.1. A preferred
MRM transition for the quantitation of PTAD-25-hydroxyvitamin D3 is
fragmenting a precursor ion with a m/z of about 558.3 to a product ion
with a m/z of about 298.1. However, as can be seen in the product ion
scans in FIGS. 11B and 12B, additional product ions may be selected to
replace or augment the preferred fragment ion.

[0162] PTAD derivatives of 1α,25-dihydroxyvitamin D2 and
1α,25-dihydroxyvitamin D3 were prepared by treating aliquots
of stock solutions of each analyte with PTAD in acetonitrile. The
derivatization reactions was allowed to proceed for approximately one
hour, and were quenched by adding water to the reaction mixture. The
derivatized analytes were then analyzed according to the LDTD-MS/MS
procedure outlined above.

[0163] Exemplary Q1 scan spectra from the analysis of samples containing
PTAD-1α,25-dihydroxyvitamin D2 and
PTAD-1α,25-hydroxyvitamin D3 are shown in FIGS. 13A, and 14A,
respectively. These spectra were collected with LDTD-MS/MS by scanning Q1
across a m/z range of about 520 to 620.

[0164] Exemplary product ion scans generated from three different
precursor ions for each of PTAD-1α,25-dihydroxyvitamin D2 and
PTAD-1α,25-hydroxyvitamin D3 are presented in FIGS. 13B-D, and
14B-D, respectively. The precursor ions selected in Q1 and the collision
energies used to generate these product ion spectra are indicated in
Table 16.

[0165] Exemplary MRM transitions for the quantitation of
PTAD-1α,25-dihydroxyvitamin D2 include fragmenting a precursor
ion with a m/z of about 550.4 to a product ion with a m/z of about 277.9;
fragmenting a precursor ion with a m/z of about 568.4 to a product ion
with a m/z of about 298.0; and fragmenting a precursor ion with a m/z of
about 586.4 to a product ion with a m/z of about 314.2. Exemplary MRM
transitions for the quantitation of PTAD-1α,25-hydroxyvitamin
D3 include fragmenting a precursor ion with a m/z of about 538.4 to
a product ion with a m/z of about 278.1; fragmenting a precursor ion with
a m/z of about 556.4 to a product ion with a m/z of about 298.0; and
fragmenting a precursor ion with a m/z of about 574.4 to a product ion
with a m/z of about 313.0. However, as can be seen in the product ion
scans in FIGS. 6B-D and 7B-D, several other product ions are generated
upon fragmentation of the precursor ions. Additional product ions may be
selected from those indicated in FIGS. 13B-D and 14B-D to replace or
augment the exemplary fragment ions.

[0167] Exemplary MRM transitions for the quantitation of
PTAD-1α,25-dihydroxyvitamin D2-[26, 26, 26, 27, 27,
27]-2H6 include fragmenting a precursor ion with a m/z of about
556.4 to a product ion with a m/z of about 278.1; fragmenting a precursor
ion with a m/z of about 574.4 to a product ion with a m/z of about 298.1;
and fragmenting a precursor ion with a m/z of about 592.4 to a product
ion with a m/z of about 313.9.

[0168] Exemplary MRM transitions for the quantitation of
PTAD-1α,25-dihydroxyvitamin D3-[6, 19, 19]-2H3
include fragmenting a precursor ion with a m/z of about 541.4 to a
product ion with a m/z of about 280.9; fragmenting a precursor ion with a
m/z of about 559.4 to a product ion with a m/z of about 301.1; and
fragmenting a precursor ion with a m/z of about 577.4 to a product ion
with a m/z of about 317.3. Exemplary MRM transitions for the quantitation
of PTAD-1α,25-dihydroxyvitamin D2-[26, 26, 26, 27, 27,
27]-2H6 include fragmenting a precursor ion with a m/z of about
544.4 to a product ion with a m/z of about 278.0; fragmenting a precursor
ion with a m/z of about 562.4 to a product ion with a m/z of about 298.2;
and fragmenting a precursor ion with a m/z of about 580.4 to a product
ion with a m/z of about 314.0.

[0169] PTAD derivatives of vitamin D2, and vitamin D3 were
prepared by treating aliquots of stock solutions of each analyte with
PTAD in acetonitrile. The derivatization reactions was allowed to proceed
for approximately one hour, and were quenched by adding water to the
reaction mixture. The derivatized analytes were then analyzed by MS/MS.

[0170] Exemplary Q1 scan spectra from the analysis of samples containing
PTAD-vitamin D2, and PTAD-vitamin D3 are shown in FIGS. 15A and
16A, respectively. These analyses were conducted by directly injecting
standard solutions containing the analyte of interest into a Finnigan TSQ
Quantum Ultra MS/MS system (Thermo Electron Corporation). A liquid
chromatography mobile phase was simulated by passing 800 μL/min of 80%
acetonitrile, 20% water with 0.1% formic acid through an HPLC column,
upstream of introduction of the analyte. The spectra were collected by
scanning Q1 across a m/z range of about 500 to 620.

[0171] Exemplary product ion scans generated from precursor ions for each
of PTAD-vitamin D2 and PTAD-vitamin D3 are presented in FIGS.
15B and 16B, respectively. The precursor ions selected in Q1 and the
collision energies used to generate these product ion spectra are
indicated in Table 17.

[0172] An exemplary MRM transition for the quantitation of PTAD-vitamin
D2 includes fragmenting a precursor ion with a m/z of about 572.2 to
a product ion with a m/z of about 297.9. An exemplary MRM transition for
the quantitation of PTAD-vitamin D3 includes fragmenting a precursor
ion with a m/z of about 560.2 to a product ion with a m/z of about 298.0.
However, as can be seen in the product ion scans in FIGS. 15B and 16B,
several other product ions are generated upon fragmentation of the
precursor ions. Additional product ions may be selected from those
indicated in FIGS. 15B and 16B to replace or augment the exemplary
fragment ions.

[0174] An exemplary MRM transition for the quantitation of PTAD-vitamin
D2-[6, 19, 19]-2H3 includes fragmenting a precursor ion
with a m/z of about 575.2 to a product ion with a m/z of about 301.0. An
exemplary MRM transition for the quantitation of PTAD-vitamin
D2-[26, 26, 26, 27, 27, 27]-2H6 includes fragmenting a
precursor ion with a m/z of about 578.2 to a product ion with a m/z of
about 297.9.

[0175] An exemplary MRM transition for the quantitation of PTAD-vitamin
D3-[6, 19, 19]-2H3 includes fragmenting a precursor ion
with a m/z of about 563.2 to a product ion with a m/z of about 301.0. An
exemplary MRM transition for the quantitation of PTAD-vitamin
D3-[26, 26, 26, 27, 27, 27]-2H6 includes fragmenting a
precursor ion with a m/z of about 566.2 to a product ion with a m/z of
about 298.0.

[0176] The contents of the articles, patents, and patent applications, and
all other documents and electronically available information mentioned or
cited herein, are hereby incorporated by reference in their entirety to
the same extent as if each individual publication was specifically and
individually indicated to be incorporated by reference. Applicants
reserve the right to physically incorporate into this application any and
all materials and information from any such articles, patents, patent
applications, or other physical and electronic documents.

[0177] The methods illustratively described herein may suitably be
practiced in the absence of any element or elements, limitation or
limitations, not specifically disclosed herein. Thus, for example, the
terms "comprising", "including," containing", etc. shall be read
expansively and without limitation. Additionally, the terms and
expressions employed herein have been used as terms of description and
not of limitation, and there is no intention in the use of such terms and
expressions of excluding any equivalents of the features shown and
described or portions thereof. It is recognized that various
modifications are possible within the scope of the invention claimed.
Thus, it should be understood that although the present invention has
been specifically disclosed by preferred embodiments and optional
features, modification and variation of the invention embodied therein
herein disclosed may be resorted to by those skilled in the art, and that
such modifications and variations are considered to be within the scope
of this invention.

[0178] The invention has been described broadly and generically herein.
Each of the narrower species and subgeneric groupings falling within the
generic disclosure also form part of the methods. This includes the
generic description of the methods with a proviso or negative limitation
removing any subject matter from the genus, regardless of whether or not
the excised material is specifically recited herein.

[0179] Other embodiments are within the following claims. In addition,
where features or aspects of the methods are described in terms of
Markush groups, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual member or
subgroup of members of the Markush group.